ANALYTICAL CHEMISTRY
754
LITERATURE CITED
While not fully investigated, it is believed that a difference in
RF values of 0.05 to 0.1 between two components would he suffi-
(1)
cient to permit a complete separation. K i t h substances whobe bands were especially diffuse the difference in RF values nould naturally have t o be larger.
(2)
(3)
(4) ACKNOWLEDGMEhT
T h e authors gratefully acknowledge the financial support of the Atomic Energy Commission. This noik was performed under Contract 8T-(30-1)-860.
(5)
(6)
Elbeih, McOmie, and Pollard, Discicssion~.Faraday SOC.,KO.7, 183 (1949). Erlenmeyer and Dahn, Helr. Chi7rz. Acta, 22, 1369 (1939) Hansen, Gunnar, Jacobs, and Simons. J . Ant. Chem. SOC., 72, 5043 (1950). Laskowski and McCrone, Abstracts of 119th Meeting, . h i , CHEM. Soc., Cleveland, Ohio, p. 19B, 1951. Robinson, Melallurgia, 37,45 (1947). Schwab and Jockers, Angezc. Chmu., 50, 546 (1937).
RECEIVED for review August 22, 1951. Accepted October 31, 1931. Contribution 839, Department of ChemiPtrj-, Vniversit)- of Pittsburgh.
Alpha-Keto Acids in Blood and Urine Studied by Paper Chromatography DiVID SELIGS0,N' AND BERN.4RD SHAPIRO George S. Cox Medical Research Institute, University of Pennsylvania, Philadelphia, Pa.
l H E accurate and specific determination of keto acids in blood and urine has been inipossihle because of the lack of bpecific, sensitive, stoichiometric reactions. Cavalliqi and coworkers (3, 4) converted the keto acids of blood and urine to their 2,4-dinitrophenylhydrazones,sepamted this group on papc'r chromatograms, and measured 'the isolated hydrazones colorimetrically. The method reported here is B modification of Cavalh i ' s , which in the author's laboratory was easier to execute. Previously described methods ( 2 , 5 , 7-13, 18) for pyruvic acid, the main a-keto acid of blood and urine, have lacked the specificity of the method mentioned above, although a n approach to specificity was made by Friedemann and Haugen ( 8 ) , \+-hotook advantage of the difference in solubilities of the dinitrophenylhydrazones. Other workers, using diatomaceous earth colunins ( 1 4 ) and alumina columns ( 6 ) , have recently reported the separation and measurement of keto acid dinitrophen>.lhydrazones. The latter method (6) is not sensitive enough for the determination of very small quantities. I n the early experiments of this laboratory, separation of keto acid hydrazones was attempted by fractionating on a silica gel column and chromatographing the fractions on paper. .4lthough this method gave a resolution of keto acids of urine, coincidental studies Ivith paper chromatography alone, which gave 'complete and specific. resolution, indicated the use of this simpler method. The usefulness of the present niethotl lies in its specificity for individual keto acids. I t has enabled the identification and quantitative estimation of two intelmedintes of carbohydrate metabolism in blood and urine nntl the demonstration of a t least two other unidentified keto acids normally present in urine. The method is reliable for samples roiitaining R S little as 5 mic.rograms or less of pyruvic or a-ketoglutaric acids. hI ETHODS
Reagents. Sulfuric acid, 0.66 S. Aqueous sodium tungstate solution, loc ;. Sodium carbonate 1 N . Sodium bicarbonate, 1 AT. Sodium hydroxide, 1 N . IIydrochloric acid, 6 1%;. Chloroform, containing 20", ethyl alcohol. Acetone. 2,4-Dinitrophenylhydrazine Solution. This contained 5 nig. of 2,l-dinitrophenylhydrazine per nil. of 6 S hydrochloric acid. T h e reagent was dissolved with gentle heating on a steam bath and constant su-irling. Solvent for Chromatography. One part of butanol and 2 parts of 1 S sodium bicarbonate were shaken in a separatory funnel for 2-minute periods several times during the course of the esperinient, to equilibrate the solvent with the water. 1
Present address, Army Medical Center, XTushington 12, D. C.
Standards. Pure sodium pyruvate and or-ketoglutaric acid were dissolved in water t o give a single solution containing 0.25 micromole per ml. of each. When 1 ml. of this standard was diluted t o 50 nil. with water, this was equivalent to 5 ml. of blood or urine containing 0.05 micromole of pyruvic acid and 0.05 micromole of a-ketoglutaric acid per nil. (or 0.44 and 0.73 mg. per 100 ml., respectively). Filter Paper for Chromatography. Strips of Whatman KO. 1 filter paper, 10 by 51 cm., were used. These were previously wetted with 1 N sodium bicarbonate ( p H 8.2) and dried. Variations in pH as well as in the concentration, cations, and anions of the buffer were tested. It was found that 1 iV sodium bicarbonate produced the best resolution of the hydrazones. The papers were hung in a trough which fitted across the top of a cylindrical jar. The jar had a rubber collar, which permitted it to be tightly covered with an appropriate enamel pan. Procedure. Ten milliliters of freshly drawn venous blood were immediately added t o 70 ml. of water and 10.0 ml. of 0.66 S sulfuric acid. This was shaken briefly, 10.0 nil. of lOyo sodium tungstate were added, and after further shaking the misture was filtered. Removal of the protein in this manner does not interfere with keto acid recoveries. I n recovery experiments the standard was added directly t o xhole blood. Standards, prepared as above, were treated like the blood filtrates. Urine, diluted 5.00 nil. t o 50 ml., was run similarly. T o 50.0 ml. of sample (blood filtrate, diluted urine, standard, or blank j 2 . 0 0 ml. of 2,4-dinitrophenylhydrazine reagent wert added. The mixture was a l l o w d t o stand for 30 minutes at 25 Cy. It was then transferred to a 125-ml. separatory funnel and the hydrazones were extracted from the aqueous solution with three 15-ml. portions of chloroform-ethyl alcohol solvent. In the case of urine i t was necessary to centrifuge for 5 minutes after extraction in order t o separate the emulsion that formed. The combined chloroform layers were extracted with 15 nil. of 1 .V sodium carbonate and the chloroform was discarded. After the sodium carbonate containing the hydrazones had been washed with 10 ml. of chloroform-ethyl alcohol, the carbonate solution cidified with 5 ml. of 6 S hydrochloric acid. The hydrazones were then extracted from the aqueous layer with three successive portions of chloroform-ethyl alcohol, 10, 5, and 5 ml., respectively. T h e three extracts containing the keto acid derivatives were combined and evaporated under a gentle air blast. Evaporation required 30 t o 45 minutes. The total residue from the chloroform-carbonate-chloroform estmction, consisting chiefly of the keto acid hydrazones, was quantitatively transferred t o paper by dissolving i t in 0.3 ml. of acetone and applying the solution to the top of the paper strip ti,ansversely as an 8-em. streak. The evaporation vessel was rinsed ivith three 0.2-ml. additional portions of acetone, each of which was applied t o the paper along the original streak. .4 gentle blast of warm air accelerated the drying. Aftel, this transfer the paper was suspended in the glass cylinder, PO t h a t the top edge dipped into the trough while the bottom edge of the strip hung 2 cm. above the bottom of the cylinder. T h e bicarbonate solution, after equilibration with butanol, was placed in the bottom of the cylinder, which was tightly covered with an enamel pan. After an equilibrium period of a half hour or more, the butanol was added t o the trough. The butanol was allowed t o move down the paper overnight (14 hours), achieving complete resolution of the keto acid hydrazones. The paper was then removed from the apparatus and air-
75s
V O L U M E 2 4 , NO. 4, A P R I L 1 9 5 2 dried. Perimeters of the keto acid hydrazone streaks were traced with pencil under ultraviolet light. The streaks were identified by RFmeasurements or by markers using pure keto acid hydrazones. RF values of a-ketoglutaric, oxalacetic, and phenylpyruvic acid hydrazones were 0.04, 0.06, and 0.91, respectively. Standard solutions of pyruvic acid yield a prominent spot at 0.62 and a much fainter one a t 0.80 (isomers?). The two spots were added t o obtain total pyruvic acid values. The 2,4-dinitrophenvlhydrazine reagent has an RF close t o 0.60, and because trace amounts of it appeared in the hydrazone residue a blank had t o be run with the pyruvic acid determinations. Each streak was cut out, sliced into small pieces, and placed in a bottle. Ten milliliters of 1 S sodium hydroxide were added t o each vessel t o elute the hydrazones. The vessels were then shaken for 10 minutes. Elution of the hydrazones from paper was found t o be quantitative. The content of each vessel I ~ filtered and the density of the red color of the filtrate was determined in a photoelectric colorimeter using a 455 filter. The rolor densities of standards of varied concentration followed the BeerLambert law.
Factors Studied to Achieve Above Conditions. Ten milligrams of 2,4-dinitrophenylhydrazine (after studying varying amounts of this reagent) appeared t.0 be sufficient for complete conversion of 0.25 micromole of a-ket,oglutaric and 0.50 micromole of pyruvic acid to their hydrazones. The chloroforni solvent' containing 20% ethyl alcohol extracted as much of the hydrazones from the reaction mixture its did chloroform containing 50% ethyl alcohol. Chloroform containing less than 20% ethyl alcohol gave less complete extractions. The three extractions with chloroform-ethyl alcohol removed :ill the pyruvic acid hydrazone and 88% of a-ketoglutaric acid. RESULTS
The recovery values obtained when a-ketoglutaric and pyruvic acids were added to blood are seen in Table I. These recoveries compare well with those reported by other methods, and indicate the validity of using tungstic acid ( I S ) as a deproteinizing agent. -~-
____._~_____~
Table I.
Recovery of Keto Acids Added to Blood or L-rine
Saingle Blood bank plasma Bank blood Bank blood (3 rno.) Diabetic blood Sornial urine Vrine frozen 4 days Bloodbankplasnia
I
Rank blood Bankblood(3mo.) Diabetic blood Normal urine Vrinefrozen4daya
Concentration, Micromole ixr~I&~Il-~Sample Total Total (observed) Added calcd. (observed) Recovery of Pyruvic Acid 0.177 0.200 0.377 0.312 0.363 0,200 0.370 0.170 0,097 0 078 0 025 0.103 0.I41 0.050 0.191 0.186 0.249 0 050 0.253 0.203 0.172 0.I26 0,060 0.186 0.114 0 077 0.040 0.111 Recovery of a-Ketoglutaric Acid 0.106 0.100 0.117 0 017 0.117 0.104 0.017 0.100 0.018 0.025 0 043 0.037 0 039 0.050 0.089 0 084 0 016 0.025 0.041 0.039 0 258 0.060 0.318 0.312 0.175 0.040 0.215 0.203
R ~ ~ ery,
"0
99 98 94 97 99 93 98
91 89 86 94 95 98 95
The reshution of blood keto acid hydrazones reveals t w o prominent streaks at RF 0.04 and 0.62 and a faint one at 0.80. The first streak was identified as a-ketoglutaric acid and t h c other two as pyruvic acid. There is another streak which rc,ninins close to the solvent front representing neutral ketones, \vhich is rejected. Resolution of urine keto acid hydrazones results in streaks a t RF 0.10 and 0,48 in addition to those of a-keto,wlutaric and pyruvic acids. The former are as yet unidentified. Osalacetic acid has not been found in blood or urine, but this \vas riot unerperted, as this cnonipound is so labile. Five pure samples of pyruvic acid from different sourws gave two streaks ( R F0.62 and 0.80) under the conditions of the reaction. Addition of these gave consistent and constant ratios of optical densit.y tQ concentration, whereas rejection of the streak a t RF 0.80 did not. These t M - 0 streaks appear to be isomers of
Table 11. Concentration of Pyruvic Acid and a-Ketoglutaric dcid in Normal Blood a-Iie t ogl utaric
Acid,
1Iicromole per M I . _-_____-
Method Range Average 0.008--0 013 O.OIOn This method Vinet and Raoul (I 7) .. Villano ( 16, 16) 0.34S10'685 ,. rriedemann (9) . . . , , Klein (IS) . ... .. Bueding a n d Wortis ( 1 ) . ... Cavallini ( 4 ) ..... 0.014e a b C
Pyruvic bcid. hlicromole per MI. Range Average 0.03043.088 0.062= 0.0680.170 0 . 1 2 3 0 068-0.159 0.061-0.109 0,088 0.08Bb 0.086-0: 140 0 . I16 .... 0 03ZC
Eight determinations on 6 subjects. Based on 20 normal blood samples. Based on 5 normal blood saniplrs.
P
Table 111. Concentrations of Alpha-Ketoglutaric Acid and Pyruvic .icid in the Blood of Patients Patient Sornial (average of 6) R . L. Xeuroblastoma w i t h n1eta.tases t o liver
A . G . Hypertensire (ane 33) L. w,Hypertensive (age 2 i ) d u r i n g cortisone c. s. Uremia T. 8. Mild Addison's disease AI. c. Diabetic, arteriorclerotir (fair control) AI. G. N i l d diabetic R . G. .Juvenile diabetic F. B. Juvenile diabetic
Alpha-Ketoglutaric Pyruvic Acid Arid Mirrumole p e r MI. Micromule p e r .Ul. 0.010 0.062
0 036 0 036 0 022
0 1%
0 014 0 016 0 014
0 085 0 05: 0 048
0 009 0.011 0.014 0.016
0 0 0 0
0 141 0 0.13
037 041 132 203
pyruvic acid dinitrophni?.lliy(lra~onewhich form in different proportions from time to time. Cavallini (4)rejects t'he srcond streak and as a result rei7ortzj considerably lower values for pyruvic acid in normal human than those reported here. His rwovery of pyruvate was also loner than that reported here. Table I1 shows the blood concentrations of a-ketoglut:iric~and pyruvic acids in normal persons as determined by the new method and by other workers using different methods. The l o w r values obtained by the presr,nt method as compared with the older methods reflect the improved specificity of thr' nt'w ~ ~ . method. Table 111 phows the keto acid I)looci levels of several l):itents ACKNOULEDGMENT
Pure sodium pyruvate and a-kctoglutaric acid were pw1)arerI and generously supplied by IT. C. Stadie. LITERXTURE CITED
Bueding, E., and \Yortis, H., J . Cliu. Invest., 21, 85 (1942). Case, E.M., Biochem. J . , 26,753 (1932). Cavallini, D., Frontaii, Y.,and Toschi, G., Nature, 163, 568 (1949). ~. .-. ,.
Ihid., 164,792 (1949). Clift, F. P.. and Cook, K. P., Biochnm. J., 26, 1788 (1932) Datta, S. P., Harris, H., and Rees, K. R., Ibid., 46,xxxvi (19501. Dische, Z., and Robbins, S . S., B i o c h a . Z.,271,304 (1934). Friedemann, T.E.,and Haugen, G. E., J . Biol. Chem., 147,415 (1943). Yriedemann. T. E.. Haugen, G. E., and Kmieciak, T. C , Ibicf., 157,673 (1945). Fromageot. C., and Desnuelle, P., Biochem. Z., 279, 174 (19.35). Hahn, A , , and Siemer, H.. 2. Biol.. 95,169 (1934). Jowett, M.,and Quastel, J. H., B i o c h a . J . , 31, 275 (1937). Klein, D., J . Bid. C'hrm., 145, 35 (1942). Le Page, G.A , Cancer Reseorch, 10,393 (1950). Villano, F.,and D'Amhrosio, L., Boll. SOC. ital. h i d . s p e r . , 22, 60 (1946). Villano, R., and Rota. L., Ihid., 22,554 (1946). Yinet, X.,and Raoul, S . . Bull. soc. chim. biol., 24,357 (1942). Westerkamp, H., Biochcm. Z., 263,239 (1933). RECEIVEDfor review M a y 18, 1951. Accepted October 18, 19.51. Presented before t h e Division of rlnalyticai Chemistry a t t h e 119th Meeting of the h f E R I C A I V CHEMICAL SOCIETY,Boston, Mass. Investigation supported b y a research grant from t h e Division of Research Grants a n d Fellowships of the Kational Institutes of Health, U. 5. Public Health Service.
